The broad objective of the proposed research is to determine how lysosome function is regulated. We will examine the degradation of glycosaminoglycans (mucopolysaccharides) in normal cultured human fibroblasts and in cells derived from patients with lysosomal enzyme defects. Degradation will also be meausred in intact isolated lysosomes whose function is stimulated by ATP and acetyl-CoA. We propose to define the precise role of acetyl-CoA and ATP in the degradative process and determine if lysosome function can be regulated by variations in cytosolic levels of these activators. Acetyl-CoA appears to stimulate lysosome function by virtue of its requirement as a cofactor for heparan sulfate degradation. We will determine the mechanism of acetyl-CoA uptake into lysosomes and the kinetics of its utilization by the enzyme acetyl-CoA: Alpha-glucosamine N-acetyltransferase. Activation of glycosaminoglycan degradation by ATP is accompanied by an acidification of the lysosome. We will determine if pH changes alone can account for the stimulation or if ATP has multiple effects on lysosome function. In addition to studying the uptake of cofactors into lysosomes and regulation of the degradative processes that occur, we plan to carry out experiments to follow the flux of catabolites out of the lysosome. We plan to monitor monosaccharide efflux to determine if it occurs by a carrier mediated process. We will also measure the extent of monosaccharide reutilization to determine if recycled sugar substrates are an important source for de novo glycosaminoglycan biosynthesis. The degradation of glycosaminoglycans involves numerous lysosomal enzymes functioning in a concerted manner. We will carry out studies to determine if those enzymes that function together are found physically associated in the lysosome. The existence of multi-enzyme complexes might reveal additional mechanisms for controlling lysosome function. Finally we will carry studies to probe the cytosolic environments of the lysosome. We will determine if lysosomes interact with cytoskeletal elements and if such an interaction has an effect on lysosome function.